The principal features of Shigella pathogenicity are its ability to invade epithelial cells and multiply within the cells. Expression of Shigella virulence is temperature regulated, the organism being invasive when grown at 37 degrees Celsius but phenotypically avirulent (non-invasive) when grown at 30 degrees Celsius. The proposed project will examine the mechanism by which Shigella regulates expression of its virulence (vir) genes in response to growth temperature. These experiments are designed to: 1) isolate lactose (lac) operon fusions to a variety of temperature regulated vir genes in Shigella flexneri; 2) isolate mutations affecting the temperature regulation of these vir genes; and, 3) elucidate the mechanism of action of the regulatory gene products. The hypothesis that Shigella vir genes constitute a regulon which is globally controlled by a regulatory mechanism responding to temperature changes will be tested. Methodology will include: generating vir::lac operon fusions by use of a lambda placMu vector; cloning of mutant and wild type regulatory genes; cloning of vir::lac fusions to isolate operator-promoter regions; and, biochemical and molecular analysis of regulatory gene products. Results of these studies will improve our understanding of the pathogenicity of Shigella and its regulation. The vir::lac fusions will also contribute to our long term objectives by serving as markers for cloning native vir genes with the goal of identifying gene products which can be tested as antigens in an anti-Shigella vaccine.